The fungi are a diverse collection of cell-wall enclosed eukaryotes either saprophytic or parasitic and may be morphologically described as yeasts, molds, mushrooms, or by other names. They are ubiquitous organisms, mostly innocuous, sometimes used for commercial purposes, and occasionally pathogenic.
The pathogenic fungi are included within the domain of medical mycology. This medical field recognizes categories of fungal pathogens (see Rippon, J. W., Medical Mycology, Saunders Co., Philadelphia, 1988, for example) including superficial, cutaneous, subcutaneous, and systemic infection. By far the most serious pathology caused by the fungi that clinicians face are the systemic infections. Deep tissue and systemic fungemia claim high mortality rates, particularly among immune-compromised populations.
Among the fungi capable of causing systemic fungemia, there is a dichotomy between the so-called "pathogenic" fungi and the "opportunistic" fungi. It is a deceptive nomenclature; the opportunists are the killers, and the pathogenic fungi are often self-limiting. The pathogenic fungi include Coccidioides immitis, Histoplasma capsulatum, Blastomyces dermatitidis, Paracoccidioides brasiliensis, and the subcutaneous pathogen, Sporothrix schenkii. The important opportunistic fungi include the Candidas--particularly C. albicans, C. tropicalis, C. parapsilosis, and Torulpsis (Candida) glabrata--Cryptococcus neoformans, members of the genus Aspergillus, and to a lesser extent, practically any fungus that can survive at host physiological temperatures.
Clinical diagnosis and treatment of systemic fungemia suffers several shortcomings compared to bacterial septicemia (which often occurs in the same immune-deficient population). First, antifungal chemotherapy is more toxic to the patient than analogous antibacterial chemotherapy. As a result, clinicians desire a more reliable demonstration of fungemia before prescribing antifungal agents. Second, fungemic patients have a poor prognosis, unless diagnosed early in infection. Third, fungi generally grow slower than the major barceremic organisms, and consequently diagnosis requiring an in vitro culture step is time consuming. And fourth, some of the fungi (again in diagnoses requiring in vitro cultivation) will not yield colonies on synthetic media for weeks, if at all. All of these factors, plus the fact that a wide array of fungi are potential systemic pathogens, point to the need for a direct method of fungal detection inclusive for virtually all fungi.
It is an aspect of the present invention to provide nucleic acid probes capable of detecting fungi.
It is another aspect of the present invention to provide nucleic acid probes which can hybridize to target regions which can be rendered accessible to probes under normal assay conditions.
It is yet another aspect to provide nucleic acid probes to fungal rRNA sequences useful as the basis for rapid diagnostic assays for assessing the presence of these organisms in a clinical sample.
While Kohne et al. (Biophysical Journal 8:1104-1118, 1968) discuss one method for preparing probes to rRNA sequences, they do not provide the teaching necessary to make probes to detect fungi.
Pace and Campbell (Journal of Bacteriology 107:543-547, 1971) discuss the homology of ribosomal ribonucleic acids from diverse bacterial species and a hybridization method for quantitating such homology levels. Similarly, Sogin, Sogin and Woese (Journal of Molecular Evolution 1:173-184, 1972) discuss the theoretical and practical aspects of using primary structural characterization of different ribosomal RNA molecules for evaluating phylogenetic relationships. Fox, Pechman and Woese (International Journal of Systematic Bacteriology 27:44-57, 1977) discuss the comparative cataloging of 16S ribosomal RNAs as an approach to prokaryotic systematics. These references, however, fail to relieve the deficiency of Kohne's teaching with respect to fungi, and in particular, do not provide specific probes useful in assays for detecting fungemia or its etiological agents, a broad spectrum of yeast and molds.
Hogan, et al (International Patent Application, Publication Number WO 88/03957) describe four putative fungal specific probes. None of them appear widely inclusive for fungi, nor are they related to the probes of the present invention.
Ribosomes are of profound importance to all organisms because they serve as the only known means of translating genetic information into cellular proteins, the main structural and catalytic elements of life. A clear manifestation of this importance is the observation that all cells have ribosomes.
Bacterial ribosomes contain three distinct RNA molecules which, at least in Escherichia coli, are referred to as 5S, 16S and 23S rRNAs. In eukaryotic organisms, there are four distinct rRNA species, generally referred to as 5S, 18S, 28S, and 5.8S. These names historically are related to the size of the RNA molecules, as determined by their sedimentation rate. In actuality, however, ribosomal RNA molecules vary substantially in size between organisms. Nonetheless, 5S, 18S, 28S, and 5.8S rRNA are commonly used as generic names for the homologous RNA molecules in any eukaryote, and this convention will be continued herein.
It is another aspect of the present invention to provide nucleic acid probes complementary to unique nucleic acid sequences within the 18S ribosomal ribonucleic acid (rRNA) of fungal pathogens.
As used herein, probe(s) refer to synthetic or biologically produced nucleic acids (DNA or RNA) which, by design or selection, contain specific nucleotide sequences that alloy them to hybridize under defined predetermined stringencies, specifically (i.e., preferentially, see next paragraph) to target nucleic acid sequences. In addition to their hybridization properties, probes also may contain certain constituents that pertain to their proper or optimal functioning under particular assay conditions. For example, probes may be modified to improve their resistance to nuclease degradation (e.g. by end capping), to carry detection ligands (e.g. fluorescein, 32-P, biotin, etc.), or to facilitate their capture onto a solid support (e.g., polydeoxyadenosine "tails"). Such modifications are elaborations on the basic probe function which is its ability to usefully discriminate between target and non-target organisms in a hybridization assay.
Hybridization traditionally is understood as the process by which, under predetermined reaction conditions, two partially or completely complementary strands of nucleic acid are allowed to come together in an antiparallel fashion (one oriented 5' to 3', the other 3' to 5') to form a double-stranded nucleic acid with specific and stable hydrogen bonds, following explicit rules pertaining to which nucleic acid bases may pair with one another. The high specificity of probes relies on the low statistical probability of unique sequences occurring at random as dictated by the multiplicative product of their individual probabilities. These concepts are well understood by those skilled in the art.
The stringency of a particular set of hybridization conditions is determined by the base composition of the probe/target duplex, as well as by the level and geometry of mispairing between the two nucleic acids.
Stringency may also be governed by such reaction parameters as the concentration and type of ionic species present in the hybridization solution, the types and concentrations of denaturing agents present, and the temperature of hybridization. Generally, as hybridization conditions become more stringent, longer probes are preferred if stable hybrids are to be formed. As a corollary, the stringency of the conditions under which a hybridization is to take place (e.g., based on the type of assay to be performed) will dictate certain characteristics of the preferred probes to be employed. Such relationships are well understood and can be readily manipulated by those skilled in the art.
As a general matter, dependent upon probe length, such persons understand stringent conditions to mean approximately 35.degree. C.-65.degree. C. in a salt solution of approximately 0.9 molar NaCl.
All references made herein are fully incorporated by reference.